Download Biology 3A Laboratory MITOSIS – Asexual Reproduction

Biology 3A Laboratory
MITOSIS – Asexual Reproduction
OBJECTIVE
•
•
•
•
•
•
To study the cell cycle and understand how, when and why cells divide.
To study and identify the major stages of cell division.
To relate the mitotic process to living organisms.
To understand the differences between plant and animal cell division.
To graphically illustrate the roles of cell division and cell elongation in root growth.
To learn how chromosomes are stained.
INTRODUCTION
Mitosis is a form of cell division that eukaryotic cells employ for growth, repair or regeneration of
somatic (body) cells. Some eukaryotic cells can even use mitosis as a form of asexual
reproduction to increase their numbers. Certain bacteria, cyanobacteria and many single-celled
organisms reproduce by another form of mitosis called binary fission. The ability of cells to
reproduce more of its own kind is a basic principle of the cell theory.
Every cell in our body has a cell cycle (Figure 1) which is divided into two main phases:
Interphase (non-dividing phase) and Mitotic phase (dividing phase). Interphase is the longest
phase of the cell cycle. It comprises about 90% of the cell cycle and is subdivided into three
phases: G1, S and G2. Most cells are arrested in the G1 phase of Interphase. During this G1
phase, cellular processes such as protein synthesis and metabolic activities occur. The S
phase of interphase is when DNA replication takes place which results in the copying of the
genetic information. The G2 (Gap) phase is another cellular growth phase in which the cell with
its copied genetic information is preparing for the mitotic phases.
Figure 1: A typical cell cycle.
The Mitotic Phase is the division of the duplicated
genetic material (karyokinesis) and can be
subdivided into four phases: prophase, metaphase,
anaphase and telophase. Prophase is longest of the
mitotic phases.
During this phase, the nucleoli
disappears, the nuclear envelope fragments and
begins to disappear. The chromosomal material
(chromatin) condenses becoming more distinct and visible as chromosomes. Each duplicated
chromosome consist of two sister chromatids that are held together at the centromere.
During metaphase, the chromosomes move to the metaphase plate and line up single file at
the center of the cell. In Anaphase, the centromeres break and the sister chromatids separate
and move toward opposite poles of the cell. After separation, each chromatid is considered a
chromosome. In Telophase, as the cell continues to elongate, the nuclear envelope and
nucleoli reappear. Cytokinesis, the division of the cytoplasm, occurs in conjunction with
1
telophase to create two separate daughter cells. In animal cells, a cleavage furrow develops to
pinch the cell into while in plants, the cell plate begins to form between the new daughter cells.
At the end of karyokinesis and cytokinesis, each daughter cell has a single nucleus with the
same number of chromosomes as the parent cell.
A. PLANT AND ANIMAL MITOSIS
Although the process of mitosis is the same for both plants and animals, there are differences in
presence or absences of certain cellular structures. Telophase and cytokinesis occur in both
plant and animal cells. However, plant cells form a cell plate during telophase, which will
eventually separate the two daughter cells. Animal cells form a cleavage furrow when the
chromosomes are near the poles. Animal cells also have centrioles (asters) which assist in the
shortening of the microtubules during anaphase.
When you are trying to locate and identify the various phases, not all of the cells will show the
“typical” stages of mitosis. During the tissue preparation procedure, thin sections are taken
through the whitefish blastula (early embryonic developmental stage) and the onion root tip in
such a way that not all of the nuclear material may be present. If you encounter such a cell that
does not contain any nuclear material, skip that cell and move on to one with distinct nuclear
material present.
Procedure A:
1. Obtain a prepared slide of an onion root tip (Allium sp.). Note: there will be several root
tips on this slide.
2. Place a piece of white paper in front of the slide in order to notice the three lightly
stained root tips.
3. Place the slide on the microscope stage and focus on the root tip under scanning power
(4X).
4. Find the rounded root cap and move to the area just above the root cap. This is the
region in the root where mitosis is occurring.
5. Identify and draw the 5 mitotic stages on your worksheet. Do not forget to label the
appropriate components.
6. Obtain a prepared slide of a whitefish blastula. Note: there will be several thin sections
through the blastula on this slide.
7. Place a piece of white paper in front of the slide in order to notice the three lightly
pinkish stained blastula sections.
8. Repeat the process as before with the onion root tip.
9. Make a note of the differences between plant and animal mitosis.
B. MITOTIC STAGE FREQUENCY
During the cell cycle, a cell will spend only a portion of its time in a particular phase. Interphase
is the longest of all the phases, while prophase is the longest of the mitotic phases. To
understand the relative time a cell spends in a particular phase, you will determine the
frequency for each of the five major phase of mitosis for your group, your cumulative lab
section’s data and finally the cumulative data for all lab sections. By increasing the sample size
(large population), this should increase the accuracy of the results.
2
The onion root tip is divided into three or more (or less) distinct regions: the region of cell
division, the region of elongation and the region of maturation. However, there is no clear
distinction between these regions. Keep in mind that the frequency of cell division will vary at
different levels and in different tissues of the root tip. Some cells may even have elongation
during interphase between successive divisions.
Procedure B:
1. Work in pairs.
2. Obtain a prepared slide of an onion root tip (Allium spp.). Note: there will be several root
tips on this slide.
3. Place the slide on the microscope stage and focus on the root tip under scanning power.
4. Find the rounded root cap and move to the area just above the root cap as before.
5. Focus on low power and scan for a section in which the chromosomes appear distinct.
6. Change to high power and focus.
7. Randomly count 100 cells and note the stage of mitosis that each cell is in. If you
cannot distinguish the stage or nothing is present, skip that cell.
NOTE: DO NOT count 100 cells for each stage. Count 100 cells altogether.
8. Make a running tally on Data Table 1 on your worksheet.
9. Calculate the frequency each stage is in by dividing the total for each stage by 100.
10. Record your groups data on Excel spreadsheet before you leave lab today.
11. Using Excel, construct a graph your data, the mean lab section data and the mean entire
Biology 3A laboratory sections’ data on the same graph.
12. Replace the slide and the microscope correctly to their proper areas.
C. Chromosome Staining Technique
In the first two portions of the lab, you identified and labeled the five stages of mitosis and
determined the relative time spent in each phase. In this section, you will learn a staining
technique used to observe chromosomes in a fresh onion root tip.
Procedure C:
1. Remove two 2 mm tips from an onion with a razor blade (be careful).
2. Add the two root tips to a small beaker and cover with 1 N HCl to hydrolyze and macerate
the tissue.
3. Place the beaker into 60C water bath for 5 – 10 minutes. Times could be longer or short
than what is given.
4. Rinse out HCl with water.
5. Place the material in a small quantity of the stain in a tightly stoppered vial. Staining may
take from a few minutes to several hours. Your instructor will divide the class into various
staining time durations (5, 10, 15, 20, and 30 minutes).
6. Rinse in several changes of tap water.
7. Squash stained materials with 45% acetic acid.
8. Cut the meristematic region of the stained root tips at 0.1 mm thick and squeeze out the
tissue with the tip of a dissecting needle.
9. Place the squeezed out material on a slide.
10. Apply a drop of Feulgen stain and a drop of 1 N HCl (1:1 ratio) for 1 – 3 seconds and
remove with a piece of blotting paper.
11. Put on a coverslip.
3
12. Making the “squash”:
a. Tap the coverslip gently with the eraser end of a pencil after fixing a corner of the
coverslip with a finger to avoid cover glass slipping.
b. Or place your thumbs on top of each other and then on top of the coverslip. Using
a rocking motion as you bear down with considerable force, squash the root tip.
13. Observe the preparation.
NOTE: the chromosomes should be bright magenta red in color whereas the cytoplasm is
colorless.
14. Show your instructor your stained slide after you have finished observing them.
4
Biology 3A Laboratory
Mitosis Worksheet
Name:
Lab Day & Time:
A. PLANT AND ANIMAL MITOSIS
1. Make representative drawings of the various distinct mitotic stages from both prepared
slides of the onion root tip and whitefish blastula. Label (at least once) in both plant and
animal cells, if present: nucleus, chromosomes, chromatid, centrioles or asters, spindle
fibers, cleavage furrow, cell plate, cytokinesis, karyokinesis.
PLANT
ANIMAL
Interphase
Prophase
Metaphase
Anaphase
Telophase
5
2. List several major differences you have observed between plant and animal mitosis.
B. MITOTIC STAGE FREQUENCY
3. Data Table 1: Individual mitotic stage frequency for onion root tip.
Stage
Interphase
Prophase
Metaphase
Anaphase
Telophase
# Observed - Tally
Total # Observed
Frequency
4. Data Table 2: Duration of mitotic stages.
Phase
Student Pair
# of
cells/stage
Duration
(min)
Lab Section
# of
cells/stage
Duration
(min)
All Sections
# of
cells/stage
Duration
(min)
Total # of nuclei
# in prophase
# in metaphase
# in anaphase
# in telophase
# in interphase
5. Estimate the duration of each stage based on your raw data, the mean lab section and the
mean data for all lab sections. The duration of each mitotic stage may be estimated using
the following equation:
Time/mitotic stage = number of cells/stage x 24 hr x 60 min
total number of cells
mitotic 1 hr
6. Using Excel, graph number of cells observed in each mitotic phase for your group’s data,
your cumulative lab section’s data and the entire cumulative data for all lab sections on the
same graph.
7. Based upon the data in Table 2 on the number of cells undergoing mitosis, what phase or
stage of active mitosis is the longest for: student pair, lab section and all sections?
8. Based upon the class data on the number of cells undergoing mitosis, what phase or stage
of active mitosis is the shortest for: student pair, lab section and all sections?
6
9. The nuclear envelope/membrane disappears during what stage of mitosis? Explain why.
10. When does the nucleolus reappear?
11. Are there any types of human cells that do not undergo mitosis after maturation? If so,
which are they?
C. Chromosome Staining Technique
12. Were you able to determine the different mitotic stages in your slide? If you could not see
any chromosomes, what should you do?
13. Data Table 3: Mitotic stage frequency for the fresh onion root tip.
Stage
Interphase
Prophase
Metaphase
Anaphase
Telophase
Total # Observed
Frequency
14. How does your data compare with section A?
15. List several other types of stains that are used to stain chromosomes?
7